WO1996007676A1 - Resine polymere pour synthese des liaisons bisulfure - Google Patents

Resine polymere pour synthese des liaisons bisulfure Download PDF

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Publication number
WO1996007676A1
WO1996007676A1 PCT/US1994/010058 US9410058W WO9607676A1 WO 1996007676 A1 WO1996007676 A1 WO 1996007676A1 US 9410058 W US9410058 W US 9410058W WO 9607676 A1 WO9607676 A1 WO 9607676A1
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Prior art keywords
resin
disulfide
copolymer
dmf
dtnb
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PCT/US1994/010058
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English (en)
Inventor
Brian R. Clark
Mohandas Pai
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Ekagen Corporation
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Priority to PCT/US1994/010058 priority Critical patent/WO1996007676A1/fr
Priority to AU78706/94A priority patent/AU7870694A/en
Publication of WO1996007676A1 publication Critical patent/WO1996007676A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups

Definitions

  • This invention relates to novel polymeric resins useful to effect the formation of peptide or protein disulfide bonds within or between molecules containing thiol groups.
  • Disulfide bond formation plays a critical role in protein folding. Disulfide bonds also serve to impart or maintain structural stability and biological activity in peptides and proteins.
  • the chemical synthesis of peptides containing one or more intramolecular disulfide bonds between pairs of cysteine residues usually requires the formation of these bonds after the peptide has been synthesized.
  • the preferred method of forming intramolecular disulfide bonds requires use of aqueous media containing mild oxidizing agents which do not also oxidize methionine, histidine, tryptophan and tyrosine residues of the peptide.
  • Air oxidation usually requires a high dilution of the peptide or protein in neutral or basic solution and a long reaction time to complete disulfide bond formation. Unlike other methods of disulfide bond formation, air oxidation yields H 2 O as a byproduct, enabling easy isolation of the peptide product.
  • Thiol-disulfide exchange reactions to form disulfide bonds between peptide cysteine pairs also require a high dilution of the peptide, but may be performed at basic, neutral or acid pH, depending upon the disulfide reagent selected.
  • disulfide interchange reaction Unlike air oxidation, the peptide product of a disulfide interchange reaction must be separated from the disulfide reagent and its reduced product.
  • a variety of disulfide reagents may be used, including oxidized glutathione and Ellman's reagent
  • the present invention provides resins having a
  • each moiety of the dithio substituent is covalently linked through a sulfur atom of the resin disulfide bond.
  • substituent is attached either directly to the resin or, in one preferred embodiment, to the distal end of an extended linker moiety the proximal end of which is linked to the resin.
  • the functional disulfide resin substituents that best serve this purpose are those that undergo thiol-disulfide interchange to yield two thiol moieties, each of which may convert to an energetically more stable thione tautomer and thus
  • the reduction of thione-generating disulfides is shown schematically in Figure 3, where Y is an imino nitrogen, a thioether sulfur, an ether oxygen or a carbon atom that is double bonded to another substituent.
  • Y and N in the first reaction scheme and the corresponding Y and C in the second may be part of a ring system.
  • the matrix M is any normally solid synthetic resin, i.e., a synthetic resin which has a melting point below that at which reactions involving the resin are effected. More specifically, the melting point of matrix M is preferably at least 200*C. A matrix resin melting point range of 150oC. to 250oC. is appropriate.
  • Specific matrix resins useful in the invention include, but are not limited to, polyolefins, polyacrylates, polymethacrylates, polystyrenes, 2 hydroxyethyl-methacrylate-ethylene dimethacrylate copolymers, styrene-divinyl benzene copolymer, styrene divinyl benzene copolymer onto which polyoxyethylene chains are grafted.
  • M may also be a natural product, e.g., cellulose, or derivatized controlled pore glass.
  • L or L 1 has at least two chemically reactive groups attached to the matrix (M) and also to each of the X or Z moieties.
  • L or L 1 which may be the same or different, are linkers effective to facilitate access of soluble reactants, such as thiols, to the disulfide bond.
  • L or L 1 may be, for example, any straight or branched alkyl chain, a polyalkylene glycol chain, a peptide, or a polyvinyl alcohol chain.
  • L in Formula I moieties, L must be branched as shown.
  • the linkers L or L 1 also may be crosslinked to provide resins having a plurality of Formula I or Formula II moieties.
  • L or L 1 may be 5 to 5000 Angstroms, preferably 20-60, in length.
  • Each of the moieties X and Z is covalently joined either directly or by the linker L to the matrix M such that,
  • the X and Z moieties may be the same or different.
  • the invention includes, but is not limited to, the following representative X or Z structures in which the functional moiety shown in the box is linked to a sulfur atom of the disulfide substituent of a matrix M:
  • a preferred disulfide substituted resin of Formula II has the formula:
  • the best mode presently known for producing the disulfide of Formula III is to couple M-L-NH 2 with 5-chloro-2-nitrobenzoic acid, followed by nucleophilic displacement of the aryl chloro group with the sulfur moiety of thiolacetic acid.
  • the acetyl group on the resulting aryl sulfur moiety is hydrolytically removed by treatment with aqueous sodium carbonate.
  • Formula III resin disulfide product is then formed by air oxidation of the resin aryl mercapto groups, as described in the examples.
  • the invention provides for a dilution effect on the solution-phase thiol contacting the resin. This occurs as a result of the reaction, for example, of a monothiol reactant with resin-bound, widely spaced disulfide substituents on the surface of the resin, thereby forming the intermediate mixed disulfide at a relatively low surface density.
  • the decreased apparent concentration of resin-bound mixed disulfide leads to a slower reaction with a second monothiol molecule in solution to form the soluble dimeric disulfide product.
  • the dithiol is effectively diluted on the resin surface (the so-called "pseudo dilution" effect). Consequently, the likelihood that the desired intramolecular disulfide will form is increased because the probability is decreased that a productive collision with a solution-phase dithiol molecule will occur to form an undesired dimeric molecule with an intermolecular disulfide bond.
  • the result is that, in comparison with air oxidation in solution, a higher concentration of a peptidedithiol can be contacted with the resin to form the desired intramolecular disulfide bond without generating a significant amount of the undesired intermolecular disulfide-bonded peptide dimer.
  • one object of the present invention is to provide solid support resins for synthesis of symmetrical intermolecular disulfides from thiols by a
  • It is another object of the present invention is to provide solid support resins for synthesis of intramolecular disulfide bonds in dithiol or oligothiol precursors with two or more thiol groups such as, for example, peptidedithiols.
  • Another object of the present invention is to provide solid support resins for synthesis of intramolecular disulfide bonds in di- or oligothiol compounds at relatively high concentrations compared with the corresponding reaction taking place wholly in solution.
  • a higher concentration of soluble reactant can be employed in the present invention because of the "pseudo dilution" effect arising when solution-phase components react with substituents present at low surface density on a solid surface.
  • Yet another object of the present invention is to provide solid support resins which permit easy separation of soluble disulfide product from insoluble, resin-bound thiol product. These products are generated during the thiol-disulfide
  • Yet another object of the present invention is to provide solid support resins which permit a nearly complete, i.e., at least 90% complete, conversion of soluble thiol to the soluble disulfide product by thiol-disulfide interchange. This is effected by employing resin-bound disulfide substituents which, when converted to their corresponding thiols during reversible thiol-disulfide interchange, undergo tautomerization to the corresponding thione derivatives. The formation of thiones thus effectively drives the interchange reaction to completion, rendering it essentially irreversible.
  • Another object of the present invention is to provide solid support resins which can generate soluble
  • disulfides from soluble thiols by thiol-disulfide interchange not only in neutral and basic aqueous solutions, but also in acidic aqueous solutions in which conversion of thiols to disulfides generally proceeds slowly.
  • the present invention possesses other objects and advantages, especially those pertaining to particular characteristics and features.
  • the resins described herein could be used to capture, isolate and recover monothiols under gentle conditions. They also could be used to prepare unsymmetrical disulfides.
  • Solid Support Resin A normally solid synthetic resin matrix having a disulfide moiety linked covalently thereto, either directly or by a linker.
  • Matrix The normally solid synthetic resin matrix of a solid support.
  • Polyhema Hydroxyethyl methacrylate-ethylene dimethacrylate copolymer. See Figure 7. (Melcor Technologies, Inc., 1016 El Camino Real, Suite 435, Sunnyvale, California 04087).
  • Macroprep CM Methacrylic acid/hydroxypropyl methacrylate- ethylene dimethacrylate copolymer. See Figure 8. (Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, California 94647).
  • the invention encompasses several different solid phase matrices each of which is attached to any one of several different linkers, L, which, in turn, is attached to any one of several disulfide substituents, -X-S-S-Z.
  • a type of matrix, linker and disulfide substituent for each of solid support disulfide resins Nos. 1-8 (see Figures 12-19) described below is listed in the Table I.
  • the first resin has a 5,5'-dithiobis (2-nitrobenzoic acid) substituent (DTNB) attached by each of its carboxyl groups via amide linkage to the distal amino groups of trifunctional Jeffamine T-403 (a polypropylene oxide-based triamine) which in turn, is attached by the remaining proximal amino group via urethane linkage to a hydroxyl group of 2-hydroxyethyl
  • DTNB 5,5'-dithiobis (2-nitrobenzoic acid) substituent
  • This resin is useful in preparing intramolecular disulfide bonds in neutral to mildly basic aqueous solutions.
  • peptidedithiols can be converted to the corresponding intramolecular peptide disulfides without contaminating the peptide solution with the dithio component and its reduced thiol moieties (see preceding diagram).
  • the trifunctional Jeffamine component allows for very high dilution of DTNB groups on the resin surface by permitting DTNB to be
  • Resin # 1 may be prepared by the sequence of 5 reactions as depicted by Figure 12.
  • the Ri enclosed within a circle in the Figure 12 diagram indicates 2-hydroxyethyl
  • -S... indicates a crosslink to a sulfur atom of a disulfide moiety of a like formula.
  • dimethacrylate copolymer (Polyhema, 2.2 mmoles of hydroxyl groups per gram of resin) was suspended in 30 ml of
  • DTNB 5,5'-dithiobis (2-nitrobenzoic acid)
  • DIPCDI diisopropylcarbodiimide
  • ninhydrin test for the presence of primary amine groups.
  • the resin-bound DTNB groups were reduced by suspending the dried DTNB resin in 10 ml of DMF and adding 1.4 gm of dithiothreitol (DTT). The suspension was mixed for two hours at room temperature
  • the DTNB resin product was examined for its capacity to convert reduced oxytocin (OxH 2 ) to oxytocin containing an intramolecular disulfide bond (Ox). Authentic OxH 2 was
  • Sample #'s 1, 2, 4 and 8 in Table II were lyophilized, reconstituted in 0.1 % trifluoroacetic acid (TFA) and examined by analytical, gradient-elution reverse phase HPLC. Elution was effected with a gradient of 0% Solvent A (0.1% TFA in water) to 60% solvent B (0.09% TFA in acetonitrile) over 30 minutes, and eluted peptides were detected by 214 nm
  • HPLC elution profiles are shown in Figure 21.
  • Table III is a display of the HPLC peak data (area and retention time) for HPLC of sample #'s 1, 2, 4 and 8 from
  • intramolecular disulfide bond formation requires use of a molar excess of resin-bound DTNB over dithiol component.
  • Peak # 1 is Ox and Peak # 3 is Ox-Ox with an HPLC retention time of 17.6 minutes. Peak # 2 does not correspond to a known form of oxytocin and may represent a truncated peptide
  • Resin #2 also has a DTNB substituent attached by both carboxyl groups to Polyhema resin.
  • the DTNB is crosslinked by intermolecular amide bonds to the distal amino group of each of two bifunctional Jeffamine EDR-148 molecules (triethylene glycol diamine) each of which is attached to the resin via a urethane linkage between the proximal amino group and a resin hydroxyl group.
  • a resin with a higher density of DTNB groups on the support may be used to convert smaller peptidedithiols to the corresponding intra-molecular peptide disulfides.
  • Resin #2 may be prepared as in the following sequence of 5 reactions as depicted in Figure 13.
  • R 1 is Polyhema resin and Jeffamine EDR-148 is triethylene glycol diamine.
  • Polyhema 50 gm of Polyhema (see example 1) was suspended in 200 ml of DMF. 40 gm of CDI was dissolved in 300 ml of DMF, and the resultant solution was added to the resin suspension. The suspension was mixed by argon sparging overnight at room temperature. The resin was filtered and washed 3 times with 250 ml of DMF. 200 ml of triethylene glycol diamine (Jeffamine EDR-148) was added to the resin, and the suspension was mixed by argon sparging overnight at room temperature. The Jeffamine resin was filtered and washed 3 times with @ 200 ml of DMF. 65 gm of DTNB and 42 gm of N-hydroxysuccinimide (NHS) were
  • DIPCDI diisopropylcarbodiimide
  • DMSO dimethylsulfoxide
  • diisopropylethylamine DIEA
  • DIEA diisopropylethylamine
  • the suspension was heated at 50° for 3 hours and then filtered, washed 3 times with DCM and 3 times with methanol and air dried.
  • a sample of the resin gave a negative ninhydrin test for primary amines.
  • 75 gm of dithiothreitol was dissolved in 300 ml of DMF, 25 ml of N-methylmorpholine was added and the solution was mixed by swirling.
  • the dried DTNB resin was added, the suspension was swirled and then left standing overnight at room temperature to effect reduction of the resin-bound DTNB groups.
  • the DTNB resin was filtered and washed 3 times with alternating washes of DMF and methanol, twice with alternating washes of DCM and methanol, three times with DCM and twice with pentane.
  • the washed resin was air dried and then vacuum dried overnight.
  • the resin was resuspended in 250 ml of DMF, and filtered air was drawn through the suspension for 72 hours at room
  • concentration of peptide in each supernatant was likewise determined spectrophotometrically by diluting 40 ⁇ l of
  • intramolecular disulfide bond in oxytocin occurs rapidly with respect to initial formation of the resin-bound mixed disulfide precursor.
  • acetonitrile were examined spectrophotometrically. 12 mg of Resin # 2 was placed in each of 4 tubes containing 400 ⁇ l of a solution with a varied amount of acetonitrile. The tubes were mixed for 2 hours at room temperature, and 50 ⁇ l of supernatant from each tube was diluted into 5 ml of distilled water. The 214 nm absorbency of each solution was measured, and the results are shown in Table VII.
  • Resin #3 is identical to Resin # 2 except that the
  • bifunctional Jeffamine EDR-148 linker is replaced by the shorter bifunctional spacer, ethylene diamine.
  • Resin #3 is prepared using Polyhema resin as depicted in Figure 14.
  • EDA resin was filtered, washed with DMF until the filtrate was negative for primary amino groups by the ninhydrin test. The resin was washed further with alternate washes of DMF and DCM, once more with DCM and then air dried and vacuum dried. The yield of EDA resin was 1.2 gm (106% of theory). 1.74 gm of DTNB was dissolved in 10 ml of DMF, 1.4 ml of DIPCDI was added, and the solution was mixed for 5 minutes at room temperature. The solution of activated DTNB was then added to the dried EDA resin, and the suspension was mixed overnight at room temperature. The resultant DTNB resin was filtered, washed alternately with DMF and DCM and then with DCM. Residual amino groups were
  • acetylated by suspending the resin in 10 ml of acetic anhydride and mixing for 3 hours at room temperature.
  • the resultant "capped” resin was filtered, washed alternately with DMF and DCM, followed by DCM alone and air dried.
  • a ninhydrin test of a sample of the dried capped DTNB resin was negative for amino groups.
  • the DTNB resin was resuspended in 8.8 ml of 0.5 M NaBH 4 in diglyme. After 1 hour at room temperature, 10 ml of DMF was added and the red suspension was mixed overnight at room temperature. The red-orange resin was filtered, washed with DMF until the filtrate was colorless and resuspended in 50 ml of 50% acetic acid.
  • the resin was filtered, washed twice with DMF, and resuspended in 20 ml of DMF containing 200 ⁇ l of DIEA. Air was drawn through the suspension overnight at room temperature to effect oxidative crosslinking of the resin-bound 5-mercapto-2-nitrobenzoyl groups to form DTNB groups.
  • the pale yellow DTNB resin was filtered, washed twice alternately with DMF and DCM, then DCM and air dried. The resin (1.2 gm) was dried in vacuuo.
  • Nitrogen and sulfur analysis of a sample of the dried DTNB resin revealed a nitrogen substitution of 4.56 millimoles per gm of resin, and a sulfur substitution of 0.29 millimoles per gm of resin. Since DTNB contains two sulfur atoms per mole, the sulfur substitution translates into a DTNB substitution of 0.15 millimoles per gm of resin. The excess nitrogen not associated with DTNB groups gives an EDA substitution of 2.1 millimoles per gm of resin which represents 100% conversion of the oxycarbonylimidazolide groups of CDI-reacted Polyhema to EDA groups.
  • Resin #4 is identical to Resin #2 except that the DTNB substituent has been replaced by a
  • Resin # 4 is prepared by the sequence of 5 reactions as depicted in Figure 15.
  • R 1 is Polyhema
  • DMT is N-(2-aminoethyl)
  • bromoacetic acid was dissolved in 15 ml of DMF, and 3.1 ml of DIPCDI was added. The solution was mixed for 15 minutes at room temperature and then added to a suspension of 5 gm of Jeffamine resin in 15 ml of DMF. The resin suspension was mixed for 2 hours at room temperature. The resulting
  • bromoacetamido resin was filtered, washed five times with alternating double washes of DCM and pentane and twice with pentane and then air and vacuum dried. A sample of the dried bromoacetamido resin gave a negative result in the ninhydrin test for primary amino groups. 1 gm of
  • 2,5-dimercapto-1,3,4-thiadiazole was dissolved in 2 ml of DMF and 350 ⁇ l of DIEA was added. After mixing for 2 minutes, this solution was added to a suspension of 1 gm of bromoacetamido resin in 2 ml of DMF. The suspension was heated overnight at 50o. The resin was filtered, washed and dried as above for the bromoacetamido resin. A sample of the
  • 5-mercapto-1,3,4-thiadiazole-2-thioglycolamido resin product was submitted for nitrogen and sulfur elemental analysis which revealed a nitrogen content of 4.04 millimoles per gm of resin and a sulfur content of 2.41 millimoles per gm of resin. From these results, it was calculated that the resin content of 5-mercapto-1,3,4-thiadiazole-2-thioglycolyl groups was 0.8 millimoles per gm of resin (66% of theory) .
  • the dried resin was suspended in DMSO, and air was drawn through the suspension for 24 hours at room temperature to oxidize the resin thiol substituents to dithio groups, yielding the
  • spectrophotometric determination of thiol groups was applied to measure OxH 2 thiol groups remaining after a varied time of exposure to the DTTG resin.
  • 500 ⁇ l of a 1 mg/ml solution of OxH 2 in 50% aqueous acetonitrile containing 0.1% TFA was added to 20 mg of DTTG resin to give a DTTG:OxH 2 molar ratio of
  • HPLC results are concordant with the results in
  • Resin # 5 has a DTNB substituent with both carboxyl groups attached in amide linkage to the ⁇ and ⁇ amino groups of lysine moieties which are amide-bonded through the lysine carboxyl group to the distal amino group of a bifunctional Jeffamine EDR-192 molecule (tetraethylene glycol diamine). The latter is linked by an amide bond between its proximal amino group and a craboxyl group on a hydrophilic methacrylate-ethylene
  • Resin #1 this favors formation of intramolecular disulfide bonds in larger peptides and proteins containing two or more thiol groups.
  • Resin # 5 is prepared by the sequence of reactions depicted in Figure 16.
  • R 2 in the Figure 16 diagram is a hydrophilic, macroporous methacrylate copolymer (Macro-Prep CM)
  • Boc is the butyloxycarbonyl protecting agent for amino groups and
  • Jeffamine EDR-192 is tetraethylene glycol diamine. At a high surface density of lysine groups, the DTNB may crosslink two resin-bound lysines, whereas at low lysine density, DTNB is mostly attached to the two amino groups on a single resin-bound lysine moiety.
  • the doubly coupled Boc-lysine resin was filtered, washed twice with DMF, alternately with methanol and DMF, twice with methanol and then air dried.
  • the Boc protecting group was removed by suspending the resin in TFA:water, 95:5 (volume:volume) for 4 hours at room
  • the resin was filtered, washed with DCM, then with 10% DIEA in DCM in order to convert the resin-bound trifluoroacetate salt of the lysine amino group to the free amine form.
  • the lysine resin was washed with DCM and pentane, air dried and then vacuum dried. 10 gm of DTNB and 6.1 gm of HOBT were dissolved in 120 ml of DMF. 6.3 ml of DIPCDI was added, and the suspension was mixed for 20 minutes at room temperature. 10 gm of lysine resin was added, and the
  • the DTNB resin product was filtered, washed twice with DMF, alternately with methanol and DMF, and resuspended in @ 20 ml of acetic anhydride to acetylate or "cap" the remaining amino groups. After filtering and washing the resin, the DTNB substituent of the "capped” resin was reduced to the 5-mercapto-2-nitrobenzoyl group (MNB) by room temperature, 4-hour treatment with a 5-fold molar excess of DTT in DMF.
  • MNB 5-mercapto-2-nitrobenzoyl group
  • the red MNB resin was filtered and washed, resuspended in DMF, and air was drawn through the suspension for 2 days to reoxidize resin-bound MNB groups to the DTNB substituent which was doubly linked to the resin.
  • the pale yellow DTNB resin (Resin # 5) was filtered, washed three times with methanol, air dried and then vacuum dried overnight. Resin # 5 was examined for its capacity to convert OxH 2 to Ox by thiol-disulfide interchange. 2 mg of authentic OxH 2 was dissolved in 1 ml of argon-sparged water.
  • Resin # 6 are shown in Table X below.
  • Resin #6 has DTNB substituents attached by both carboxyl groups in amide linkage to the ⁇ and ⁇ amino groups of resin lysine substituents. Lysine is amide-bonded via its carboxyl chain which is linked to a styrene-divinylbenzene copolymer (NOVASYN-TG, NovaBiochem). This resin is similar to Resin #1 and #5 in its applications involving larger peptides and proteins.
  • Resin #6 is prepared is prepared by the reactions depicted in Figure 17.
  • R 3 in the Figure 17 diagram consists of ⁇ - aminopolyoxyethylene chains grafted onto a crosslinked
  • the Fmoc protecting groups were removed by suspending the bisFmoc-lysine resin in 10 ml of 20 volume % piperidine in DCM and mixing the suspension for 30 minutes at room temperature. The resultant lysine resin was filtered, washed twice with alternate DCM and methanol washes and then twice with methanol. The resin was air dried, and a sample of the resin gave a positive ninhydrin test result, indicating removal of the Fmoc protecting group. 475 mg of DTNB and 199 mg of HOBT were dissolved in 7 ml of DMF, and 203 ⁇ l of DIPCDI was added.
  • the DTNB resin was filtered and washed with DMF. A sample of the resin gave a negative result in the ninhydrin test. The resin was further washed, twice with alternating washes of methanol and DMF and twice with methanol, and then air dried. The DTNB resin was resuspended in 5 ml of DMF, 370 mg of DTT was added, and the red suspension was mixed for 2 hours at room temperature. The resultant MNB resin was
  • a basic DMF solution was prepared by shaking 0.5 ml water and 5 gm NaOH with 50 ml DMF. 5 ml of the supernatant of this suspension was added to the MNB resin, and the suspension was mixed for 10 minutes at room temperature. 254 mg of iodine was dissolved in 1 ml of the basic DMF solution, and 110 ⁇ l of the resultant solution was added to the resin suspension.
  • the resin turned light yellow in color, indicating reoxidation of the resin-bound MNB groups to DTNB substituents.
  • the resin was filtered and washed twice with DMF, twice with methanol and then twice with DMF.
  • the slightly red resin was resuspended in DMF and 20 ⁇ l of the iodine solution was added.
  • the resin again became pale yellow in color and remained so after washing twice with DMF and twice with methanol.
  • the resin was dried, and a sample was treated with DMF containing DTT.
  • the pale yellow resin (Resin # 6) turned dark brick red with no color appearing in the
  • Resin # 6 was examined for its capacity to convert OxH 2 to Ox. 1.6 mg of OxH 2 was dissolved in 200 ml of degassed water, and 100 ml of this solution was mixed with each of two 5 mg samples of Resin # 6. After 5 minutes, an aliquot of
  • Resin #7 has a DTNB group, the two carboxyl groups of which are amide-bonded to the ⁇ and ⁇ amino groups of lysine substituents on the resin.
  • Each lysine molecule is amide- linked through its carboxyl group to an aminomethyl group which is attached to an aryl moiety of a styrene-divinylbenzene copolymer.
  • This resin is similar to the DTNB-substituted resins in application.
  • Resin #7 is prepared as in the sequence of reactions as depicted by Figure 18.
  • R 4 is styrene-divinylbenzene copolymer containing aminomethyl groups attached to aryl moieties of the resin.
  • -S... indicates a crosslink to a sulfur atom of a disulfide moiety of a like formula.
  • the resin was recoupled with bisFmoc-lysine as described above, except that the reaction was run overnight at room temperature. The resin was washed and dried as described above. A sample of the bisFmoc-lysine resin gave a negative ninhydrin test result. The Fmoc protecting group was removed from the lysine amino groups by adding the resin to 25 ml of 20 volume % piperidine in DCM and mixing the suspension for 1 hour at room
  • the deprotected lysine resin was filtered, washed twice with alternating washes of DCM and methanol and twice with DCM and then air dried. 443 mg of DTNB and 343 mg of HOBT were dissolved in 10 ml of NMP, and 350 ⁇ l of DIPCDI was added. The deprotected lysine resin was added, and the suspension was mixed overnight at room temperature. The resultant DTNB resin was filtered, washed twice with NMP, twice with methanol, twice with DCM and twice with alternating single washes of NMP and double washes of DCM. A ninhydrin test of a sample of the resin gave a positive result. The resin was recoupled
  • NaOH-saturated DMF prepared by shaking 1 gm of NaOH with a solution of 1% water in DMF
  • the suspension was mixed for 30 minutes at room temperature.
  • 25 ⁇ l of a 1 M iodine in DMF was added to reoxidize the MNB to DTNB, and the suspension was mixed for 15 minutes at room temperature during which time the resin color changed from red to dull yellow.
  • the DTNB resin was washed three times with alternating washes of DMF and methanol, once with an alternating wash of DCM and methanol and three times with DCM, and then air dried.
  • Resin #8 is identical with Resin #7 except that the DTNB group is replaced by a dithiodinicotinic acid (DTDNA)
  • Resin #8 is synthesized as shown in the sequence of
  • Aminomethyl polystyrene is styrene-divinylbenzene copolymer containing aryl aminomethyl moieties
  • DTDNA is 6, 6 '-dithiodinicotinic acid.
  • the resin was resuspended in 25 ml DMF, 1.3 gm of bisFmoc-lysine and 0.4 ml DIPCDI were added, and the suspension was mixed overnight at room temperature. The resin was filtered, washed twice with alternate washes of DMF and methanol and air dried. The bisFmoc-lysine resin was
  • DTDNA 6,6'-dithiodinicotinic acid
  • 20 ml of warm DMF 0.7 ml of DIPCDI was added, the solution was mixed and added to the lysine resin and the suspension was mixed overnight at room temperature.
  • the resultant DTDNA resin was filtered, washed twice with an alternating double wash of DMF and a single wash of methanol, then once more with methanol, and air dried.
  • a sample of the pale yellow resin gave a negative ninhydrin test result, indicating complete coupling of the lysine amino groups to DTDNA.
  • a sample of the resin was resuspended in 1 ml of DMF, and a small amount of DTT was added. Both the supernatant and the resin became bright yellow, indicating reduction of resin-bound DTDNA to
  • 6-mercaptonicotinic acid moieties of which one was released to the medium and one remained attached to the resin.
  • 1 gm of the DTDNA resin was resuspended in 10 ml of a solution of 1.6 gm of N-acetylcysteine in DMF in order to reduce the DTDNA

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Abstract

Compositions à base de résine de bisulfure dans lesquelles, suite à un échange bisulfure/thiol chaque fraction thiol reste fixée à la résine, une telle résine affectant la formation de liaisons bisulfure de protéine ou de peptide inter ou intramoléculaire.
PCT/US1994/010058 1994-09-06 1994-09-06 Resine polymere pour synthese des liaisons bisulfure WO1996007676A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1994/010058 WO1996007676A1 (fr) 1994-09-06 1994-09-06 Resine polymere pour synthese des liaisons bisulfure
AU78706/94A AU7870694A (en) 1994-09-06 1994-09-06 Polymeric resin for disulfide bond synthesis

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Application Number Priority Date Filing Date Title
PCT/US1994/010058 WO1996007676A1 (fr) 1994-09-06 1994-09-06 Resine polymere pour synthese des liaisons bisulfure

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WO1996007676A1 true WO1996007676A1 (fr) 1996-03-14

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PCT/US1994/010058 WO1996007676A1 (fr) 1994-09-06 1994-09-06 Resine polymere pour synthese des liaisons bisulfure

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AU (1) AU7870694A (fr)
WO (1) WO1996007676A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2474057A (en) * 2009-10-02 2011-04-06 Givaudan Sa A method of isolating and recovering thiol-containing compounds
US8980238B2 (en) 2009-09-30 2015-03-17 Thiomatrix Forschungs—Und Beratungs GmbH Mucoadhesive polymers having vitamin B partial structures
WO2022227927A1 (fr) * 2021-04-30 2022-11-03 浙江大学 Matériau polymère dégradable et nanocomposite auto-assemblé et son application

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US4546070A (en) * 1983-12-12 1985-10-08 Fuji Photo Film Co., Ltd. Method for processing color photographic light-sensitive material
JPS62148949A (ja) * 1985-08-30 1987-07-02 Fuji Photo Film Co Ltd ハロゲン化銀写真感光材料

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US4546070A (en) * 1983-12-12 1985-10-08 Fuji Photo Film Co., Ltd. Method for processing color photographic light-sensitive material
JPS62148949A (ja) * 1985-08-30 1987-07-02 Fuji Photo Film Co Ltd ハロゲン化銀写真感光材料

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ANALYTICAL BIOCHEMISTRY, Vol. 134, (1983), HARRIS et al., "Polyacrylamide Gels Which Contain a Novel Mixed Disulfide Compound Can Be Used to Direct Ensymes That Catalyze Thiol-Producing Reactions", pages 126-132. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8980238B2 (en) 2009-09-30 2015-03-17 Thiomatrix Forschungs—Und Beratungs GmbH Mucoadhesive polymers having vitamin B partial structures
EP2482852B1 (fr) * 2009-09-30 2017-11-22 ThioMatrix Forschungs- und Beratungs GmbH Polymères muco-adhésifs comprenant des structures partielles de vitamine b
US10639377B2 (en) 2009-09-30 2020-05-05 Thiomatrix Forschungs-Und Beratungs Gmbh Mucoadhesive polymers having vitamin B partial structures
GB2474057A (en) * 2009-10-02 2011-04-06 Givaudan Sa A method of isolating and recovering thiol-containing compounds
WO2022227927A1 (fr) * 2021-04-30 2022-11-03 浙江大学 Matériau polymère dégradable et nanocomposite auto-assemblé et son application

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